Vibrio cholerae Cytolysin (VCC) is a potent pore-forming toxin (PFT) that attacks human cells, although the specific targets of the toxin and role in pathogenesis are unknown. Understanding how pathogenic virulence factors recognize host membranes is an important first step towards developing therapies to block their action. This knowledge may also benefit the development of drugs that target diseased cells, such as cancer or virally infected cells. The long-term goal is to elucidate how pore-forming toxins recognize, assemble on, and disrupt cellular membranes from a structural and mechanistic perspective. The overall objective of this application, which is the next step towards obtaining this goal, is t understand how VCC targets motifs on cell membranes, including carbohydrate and cholesterol molecules. The VCC toxin contains two structural domains with folds similar to sugar-binding lectin proteins. Similar carbohydrate-binding domains are found on a variety of VCC toxin homologs with important human health implications, including toxins produced by Vibrio vulnificus, a cause of deadly food poisoning and sepsis. The central hypothesis of this project is that VCC recognizes membranes in a specific and reversible manner. This involves binding sites on the toxin molecule that increase the local concentration of toxin on the surface of the membrane. The rationale for the proposed work is that structural and functional characterization of VCC binding motifs will provide a clear understanding of the contribution that recognition sites make towards the picomolar cytolytic activity of the toxin. In order to accomplish this goal, three specific aims will be pursued: 1) Identify the carbohydrate ligands that VCC accessory domains target on cell membranes; 2) Structurally characterize interactions between VCC accessory domains and their respective ligands to understand the mechanism of ligand selectivity; and 3) Investigate the mechanism by which VCC recognizes cholesterol in eukaryotic membranes. Under the first aim, a combination of glycan screening and biophysical characterization will be used to identify and quantify carbohydrate ligands recog nized by VCC. Under the second aim, high-resolution structural analysis by X-ray crystallography will be used to understand the nature of ligand selectivity. Under the third aim, putative cholesterol binding-sites will be ana lyzed fo their contribution towards the recognition of susceptible cell membranes by VCC. The approach is in novative because it combines a comprehensive structural analysis with new screening technologies to answer fundamental questions about biological function. The proposed research is significant, because it will illuminate how this potent channel-forming toxin utilizes receptorsto assemble on cell membranes with high-affinity. This information will inform efforts to target toxins and drugs to specific cell membranes in order to treat infectious diseases and cancer. PUBLIC HEALTH RELEVANCE: The proposed research is relevant to public health because PFTs, such as VCC, are involved in the process of pathogen colonization and host damage. Understanding how toxins attack cells is essential for developing strategies to combat infectious disease. Therefore, the proposed research is relevant to NIH's mission to improve the health of the Nation by supporting research in the causes and cure of human diseases.